The increasing worldwide incidence of tuberculosis, which is the leading cause of death from a single infectious disease, and its frequent association with HIV-1 infection have rendered current laboratory diagnostic methods inadequate. Microscopic examination of sputum has low sensitivity, and culture techniques are too slow. DNA sequence amplification by PCR is sufficiently sensitive only in patients with multibacillary pulmonary TB. New diagnostic methods are urgently needed. The PI believes that serology presents an attractive approach. Serodiagnostic tests are simple, rapid, inexpensive and do not require the presence of mycobacteria in the clinical specimens. The present lack of a highly accurate immunoassay for TV is primary due to insufficient knowledge of the humoral immune response to TV and to a limited availability of purified mycobacterial antigens. During the current funding period, the PI developed a panel of eleven recombinant, highly purified antigens of M. tuberculosis and found a remarkable patient-to-patient variation in antigen recognition. The PI has also developed a strategy for multi-antigen-based serodiagnosis of TB. Antigens will be selected for a multi-antigen cocktail for diagnosis of active TB in the general population by identifying antigens that are highly seroactive and that are recognized by sera from patients with paucibacillary and multibacillary pulmonary TB. To minimize false positive results due to immune responses that are not associated with active TB, antigens for the cocktail will be chosen from those that are preferentially recognized by active TB patients rather than asymptomatic tuberculin reactors, BCG vaccines, and individuals with inactive TB. Cocktail development will be pursued by assessing the behavior of selected antigens when combined in solid phase. Peptides displaying immunodominant, M. tuberculosis complex-specific B cell epitopes on highly seroreactive proteins will be utilized to minimize antigen interference or loss of assay specificity that may be observed when antigens are combined. Special emphasis will be placed on development of diagnostic assays for TV in children and in HIV-1-infected individuals. Because of the rapid progression from infection to active TB in children, antigens recognized by serum antibodies in pediatric TB may not be the same as those recognized by the adult populations. The PI will select antigens recognized by sera from children with TB to develop serodiagnostic assays specific for pediatric TB. Another high risk group for rapid disease progression and high TB-associated mortality is the HIV-1-infected population. The PI's studies indicate that, although the overall antibody response is decreased in HIV+ TB, recognition os certain antigens of M. tuberculosis is unaltered in the presence of concomitant HIV-infection. Based on these findings, the PI proposed to develop serodiagnostic assays that meet the special needs of this population. The PI also anticipates that they will be able to identify early serological markers of TB infection in household contacts of patients with active TB.